Single-Objective Light-Sheet Microscopy for Fast 3D Super-Resolution
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Solution Overview
Problem
Traditional optical microscopy is limited by the diffraction limit, leading to challenges in observing fine biological activities, and existing super-resolution microscopy techniques like STED, single-molecule localization, and SIM have limitations such as photobleaching, phototoxicity, or reduced time resolution, while light-sheet fluorescence microscopy faces mechanical and resolution constraints.
Innovation Solution
Combining single-objective light-sheet microscopy with structured illumination super-resolution microscopy to create a system that uses a single objective for both excitation and detection, generating structured light stripes through interference of two light sheets, allowing for high-resolution, high-speed three-dimensional live imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional optical microscopy is used, then the system is simple and easy to operate, but the resolution is limited by the diffraction limit
Solution Approach 1:
The patent combines light-sheet fluorescence microscopy with structured illumination microscopy by integrating the light-sheet generation and phase adjustment module into the existing microscopy system. This merging allows the system to achieve super-resolution capabilities while maintaining the simplicity of light-sheet illumination, resolving the contradiction between improved resolution and increased system complexity.
Solution Approach 2:
The single objective lens serves multiple functions: it acts as both the excitation objective for light-sheet illumination and the detection objective for fluorescence signal collection. This multi-functionality simplifies the overall system structure while achieving super-resolution, addressing the contradiction between resolution improvement and system complexity.
2Measurement precision
If STED microscopy technology is used, then super-resolution is achieved, but photobleaching and phototoxicity increase
Solution Approach 1:
The patent segments the illumination into a thin light-sheet that selectively excites only the focal plane of the sample, rather than illuminating the entire sample volume. This segmentation reduces the total light dose and eliminates out-of-focus photobleaching, achieving super-resolution while minimizing photobleaching and phototoxicity.
Solution Approach 2:
The light-sheet illumination provides localized excitation only at the focal plane where the sample is positioned, while the detection objective collects fluorescence from the same localized region. This local quality approach ensures that only the necessary portion of the sample receives excitation light, reducing overall photobleaching and phototoxicity while maintaining super-resolution.
3Measurement precision
If single-molecule localization microscopy is used, then spatial resolution is improved, but time resolution is sacrificed
Solution Approach 1:
The patent employs continuous light-sheet illumination combined with structured illumination patterns that can be rapidly switched and scanned across the sample. This continuous action allows for high-speed imaging at super-resolution, eliminating the need to sacrifice time resolution for spatial resolution as in single-molecule localization methods.
4Productivity
If SIM technology is used, then high-speed live imaging is enabled, but photobleaching and reconstruction artifacts increase
Solution Approach 1:
The patent segments the illumination into a thin light-sheet that selectively excites only the focal plane, eliminating out-of-focus fluorescence that causes reconstruction artifacts in SIM. This segmentation also reduces total photobleaching by limiting excitation to only the necessary region, while maintaining high-speed imaging capability.
5Object-affected harmful factors
If traditional light-sheet microscopy is used, then photobleaching is reduced, but the system cannot accommodate standard slides and multi-well plates
Solution Approach 1:
The patent designs the light-sheet generation and phase adjustment module to work with standard microscope configurations, including standard slides and multi-well plates. The single objective lens and remote focusing mechanism provide universal applicability across different sample types while maintaining the photobleaching reduction benefits of light-sheet illumination.
6Productivity
If light-sheet fluorescence microscopy is used, then long-term and high-speed imaging is achieved, but spatial resolution is not very high
Solution Approach 1:
The patent merges light-sheet fluorescence microscopy with structured illumination microscopy by integrating the light-sheet generation and phase adjustment module into the microscopy system. This combination achieves both high-speed imaging and super-resolution, resolving the contradiction between imaging speed and spatial resolution.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The system achieves super-resolution in three dimensions with reduced photobleaching and reconstruction artifacts, suitable for standard slides and multi-well plates, facilitating high-speed imaging of cells, tissues, and embryos.
Implementation Method 1
output two light sheets, a scanning module, configured to guide the two light sheets outputted by the light sheet generation and phase adjustment module to a single objective, the single objective, arranged downstream of the scanning module, and configured to allow the guided two light sheets to pass therethrough and interfere with each other to form a structured light stripe region
Implementation Method 2
receive fluorescence signals, a fluorescence detection module, configured to record the fluorescence signal received by the single objective
Data Source
AI summary
A super-resolution single-objective light-sheet optical microscopy system and method, and a related imaging system, are configured to generate two light sheets that are guided to a single objective. The single objective is configured to allow the two light sheets to transmit through same and interfere with each other to generate structured light fringe regions. The single objective is further configured to receive a fluorescence signal that is recorded by a fluorescence detection module.